Fish collagen peptide with skin brightening and freckle removing effects as well as preparation method and application of fish collagen peptide

Abstract

The invention provides a fish collagen peptide with skin brightening and freckle removing effects as well as a preparation method and application thereof, and belongs to the technical field of protein peptides. The preparation method comprises the following steps: by taking fish skin and / or fish scales as raw materials, performing degreasing and fishy smell removal treatment, and then sequentially performing heat treatment, homogenization treatment, enzymolysis treatment and enzyme deactivation refining to obtain the fish collagen peptide. In the fish collagen peptide prepared by the invention, the proportion of polypeptide with the molecular weight of 189-3000Da is more than 90%, and the protein content is more than 99.23%. Experiments show that the fish collagen peptide can effectively reduce the skin color spot area proportion of a subject and improve the skin L value and ITA value of the subject at the same time. Therefore, the fish collagen peptide prepared by the invention has good skin brightening and freckle removing effects, and a new way is provided for preparing related products.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein peptide preparation, and specifically relates to a fish collagen peptide with skin brightening and freckle removing effects, and a preparation method and application thereof. Background Art

[0002] Collagen mainly exists in human skin, bones, eyes, teeth, tendons, internal organs (including heart, stomach, intestines, blood vessels), etc. Its function is to maintain the shape and structure of the skin and tissue organs, and it is also an important raw material for repairing damaged tissues. Among the components of human skin, 70% is composed of collagen. When there is insufficient collagen, problems are likely to occur in the skin. When the collagen in the dermis is oxidized and broken, the supporting effect on the epidermis disappears, resulting in uneven collapse, which forms wrinkles. Now, collagen is slowly entering the beauty and skin care field to reduce the loss of collagen by supplementing collagen in vitro.

[0003] Fish skin and fish scales are rich in collagen. Therefore, fish skin and fish scales are often used to prepare fish collagen peptides. Currently, the collagen peptides prepared by the preparation methods of fish collagen peptides are used for anti-aging, improving skin moisture retention and reducing wrinkles. After retrieval, there is no report on the preparation method of fish collagen peptides with skin brightening and freckle removing functions. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a preparation method of fish collagen peptides, so that the prepared fish collagen peptides have good skin brightening and freckle removing effects.

[0005] The present invention provides a preparation method of fish collagen peptides, comprising the following steps: Performing heat treatment and homogenization treatment on defatted and deodorized fish scales to obtain a pretreated material; The method for defatting and deodorizing the fish scales includes soaking treatment with an oxidant and an acid or a base simultaneously; Performing protease hydrolysis on the pretreated material under vortex conditions, and after inactivating the enzyme, obtaining an enzymatic hydrolysate; The protease includes at least two of alkaline protease, neutral protease, bromelain and papain; Separating the liquid phase from the enzymatic hydrolysate and removing impurities to obtain fish collagen peptides.

[0006] Preferably, the pH value of the defatted and deodorized fish scales is 6.5 - 7.5; The conductivity of the defatted and deodorized fish scales < 500 μS / cm.

[0007] Preferably, the oxidant includes an oxidant with any of the following working concentrations: chlorine dioxide preparation with a volume concentration of 0.6% - 2.0%, hydrogen peroxide with a volume concentration of 0.1% - 0.5%, and ozone with a concentration of 0.1 - 0.15 ppm; The acid includes an aqueous citric acid solution or an aqueous malic acid solution with a working concentration of 0.9% - 1.5% by mass, or an aqueous hydrochloric acid solution with a working concentration of 0.1% - 0.3% by volume; The base includes an aqueous sodium hydroxide solution with a working concentration of 0.1% - 0.3% by mass.

[0008] Preferably, the temperature of the heat treatment is 55 - 100 °C; the time of the heat treatment is 30 - 90 min.

[0009] Preferably, the homogeneity treatment includes high-speed shearing, emulsification, homogenization, and homogenizing; The rotation speed of the high-speed shearing is 10000 - 18000 rpm; the time of the high-speed shearing is 30 - 60 s; The speed of the emulsification is 2000 - 2500 r / min, and the time of the emulsification is 20 - 30 min; The rotation speed of the homogenization is 20000 - 25000 r / min, and the time of the homogenization is 30 - 60 s; The pressure of the homogenizing is 1000 - 1500 bar, with two-stage homogenization.

[0010] Preferably, in the enzymatic hydrolysis system, the mass percentage of the alkaline protease is 0.1‰ - 1.5‰; The mass percentage of the neutral protease is 0.4‰ - 1.2‰; The mass percentage of the bromelain is 0.1% - 0.5%; The mass percentage of the papain is 0.3‰ - 1.0‰; The temperature of the enzymatic hydrolysis by the protease is 55 - 65 °C; The pH value of the system during the enzymatic hydrolysis by the protease is 7.0 - 8.5; The reaction time of the enzymatic hydrolysis by the protease is 1 - 3 h.

[0011] Preferably, the impurity removal includes decolorization and re-deodorization; The decolorization includes decolorization by membrane filtration; the pore size of the membrane is 5000 - 20000 Da; The method of the re-deodorization includes activated carbon adsorption.

[0012] The present invention provides a fish collagen peptide prepared by the said preparation method. In the fish collagen peptide, the mass percentage of peptides with a molecular weight greater than 10,000 Da is 0.01% - 0.02%, the mass percentage of peptides with a molecular weight of 5,000 - 10,000 Da is 0.04% - 0.83%, the mass percentage of peptides with a molecular weight of 3,000 - 5,000 Da is 0.68 - 4.58%, the mass percentage of peptides with a molecular weight of 1,000 - 3,000 Da is 13.82% - 34.42%; the mass percentage of peptides with a molecular weight of 500 - 1,000 Da is 31.96% - 32.76%, the mass percentage of peptides with a molecular weight of 189 - 500 Da is 24.15% - 47.42%, and the mass percentage of peptides with a molecular weight less than 189 Da is 3.24% - 5.89%.

[0013] The present invention provides the application of the said fish collagen peptide in the preparation of drugs or skin care products for brightening skin and removing freckles, or in the preparation of health products, foods or food additives.

[0014] The present invention provides a preparation method of fish collagen peptide, which includes the following steps: performing heat treatment and homogenization treatment on degreased and deodorized fish scales to obtain a pretreated material; the method for degreasing and deodorizing the fish scales includes soaking treatment with an oxidant and an acid or a base simultaneously; performing protease hydrolysis on the pretreated material under vortex conditions, and after inactivating the enzyme, obtaining an enzymolysis product; the protease includes at least two of alkaline protease, neutral protease, bromelain and papain; separating the liquid phase from the enzymolysis product and removing impurities to obtain fish collagen peptide. Compared with the degreasing and deodorizing treatment using an oxidant or an acid or a base alone, the simultaneous soaking treatment with an oxidant and an acid or a base in the present invention plays a synergistic role, which can greatly reduce the content of lipid components in fish scales, making the prepared fish collagen peptide free of detected fat, and at the same time ensuring that the prepared product has no fishy smell, improving the consumer experience. At the same time, the fish collagen peptide prepared by the method of the present invention has been clinically verified to have a good effect of brightening skin and removing freckles. After 28 days of testing on the subjects, the results show that the freckle area has decreased by 15.34% compared with that before the test, showing a significant difference; at the same time, from the test results of the skin L value, it can be known that the skin L value has increased by 2.19% compared with that before the test, and there is a significant difference; the present invention also tests the skin ITA value of the subjects after 28 days, and the results show that the skin ITA value has increased by 9.19% compared with that before the test, showing a significant difference. It can be seen that the fish collagen peptide prepared by the method of the present invention has the effects of improving skin brightness and removing skin freckles, and can be used in the preparation of products for brightening skin and removing freckles. Description of the Drawings

[0015] Figure 1 Measurement results of the proportion of the freckle area of the subjects before and after taking fish collagen peptide; Note: ## indicates a significant difference between the two groups P <0.05; Figure 2Determination results of skin L value of subjects before and after taking fish collagen peptide; Note: ## indicates significant difference between the two groups P <0.05; Figure 3 Determination results of skin ITA value of subjects before and after taking fish collagen peptide; Note: ## indicates significant difference between the two groups P <0.05. Specific implementation manner

[0016] The present invention provides a preparation method of fish collagen peptide, comprising the following steps: Performing heat treatment and homogenization treatment on defatted and deodorized fish scales and / or fish skins to obtain pretreated materials; Performing protease enzymolysis on the pretreated materials under vortex conditions, and after inactivating the enzyme, obtaining an enzymolysis product; The protease includes at least two of alkaline protease, neutral protease, bromelain and papain; Separating the liquid phase from the enzymolysis product, removing impurities, and obtaining fish collagen peptide.

[0017] The present invention performs heat treatment and homogenization treatment on defatted and deodorized fish scales and / or fish skins to obtain pretreated materials.

[0018] In the present invention, the fish scales or fish skins preferably come from freshwater fish or seawater fish. The source fish species of the fish scales include at least one of tilapia, grass carp, and snakehead. In specific embodiments of the present invention, the method for preparing fish collagen peptide is described by taking grass carp scales and snakehead scales as examples. The source fish species of the fish skins preferably include at least one of cod, tilapia, and basa. The fish scales or fish skins include fresh fish scales or fish skins, dried fish scales or fish skins, or frozen fish skins or fish scales. The fish scales or fish skins preferably include rinsing to remove impurities, blood, etc. The rinsing method preferably uses 2-4 times of water to wash the fish skins or fish scales, and the number of washing times is based on the washing water being clear, without obvious blood or visible impurities to the naked eye. When the fish scales or fish skins are dried raw materials, it is preferably pre-soaked and rehydrated for 12-15h, which can be 13-14h. When the fish scales or fish skins are frozen fish skins or fish scales, it is preferably pre-soaked and thawed for 18-24h, and can also be 20-22h.

[0019] In the present invention, the method for degreasing and deodorizing fish scales and / or fish skin preferably includes sequentially treating with an oxidant and soaking with an acid or a base. The oxidant preferably includes an oxidant with any of the following working concentrations: chlorine dioxide preparation with a volume concentration of 0.6% - 2.0%, hydrogen peroxide with a volume concentration of 0.1% - 0.5%, and ozone with a concentration of 0.1 - 0.15 ppm; it can be chlorine dioxide preparation with a volume concentration of 0.8% - 1.0%, hydrogen peroxide with a volume concentration of 0.2% - 0.4%, and ozone with a concentration of 0.12 - 0.14 ppm. In order to further improve the healthiness of the product, the oxidant adopts a food-grade preparation product. The acid includes an aqueous solution of citric acid or malic acid with a working concentration of 0.9% - 1.5% by mass or an aqueous solution of hydrochloric acid with a working concentration of 0.1% - 0.3% by volume; it can be an aqueous solution of citric acid or malic acid with a mass percentage of 1.2% or an aqueous solution of hydrochloric acid with a working concentration of 0.2% by volume. The base includes an aqueous solution of sodium hydroxide with a working concentration of 0.1% - 0.3% by mass; it can be 0.2%. When degreasing and deodorizing, stirring is preferably accompanied. The conditions for stirring are stirring for 2 - 5 minutes every 2 - 6 hours. After degreasing and deodorizing, it is preferred to rinse the degreased and deodorized fish scales and / or fish skin with water. After rinsing, the pH value of the degreased and deodorized fish scales and / or fish skin is 6.5 - 7.5; it can be 6.8 - 7.2, and can also be 7.0. The conductivity of the degreased and deodorized fish scales and / or fish skin is < 500 μS / cm; it can also be 386 - 479 μS / cm.

[0020] In the present invention, the temperature of the heat treatment is preferably 55 - 100 °C; it can be 58 - 80 °C, and can also be 60 - 68 °C; the time of the heat treatment is preferably 30 - 90 minutes; it can be 40 - 80 minutes, and can also be 60 - 70 minutes. The heat treatment moderately softens the fish scales, facilitating subsequent processing. The uniformity treatment preferably includes high-speed shearing, emulsification, homogenization, and homogenizing. The rotation speed of the high-speed shearing preferably includes 10000 - 18000 rpm; it can be 15000 rpm; the time of the high-speed shearing is preferably 30 - 60 seconds; it can be 45 seconds; the rotation speed of the emulsification is preferably 2200 - 2400 r / min; it can be 2400 r / min; the time of the emulsification is preferably 20 - 30 minutes; it can be 25 minutes; the conditions for homogenization are preferably 22000 - 24000 r / min; it can be 24000 r / min; the time of homogenization is preferably 35 - 45 seconds; it can be 35 seconds; the conditions for homogenizing are preferably 1200 - 1400 Bar; it can be 1300 - 1400 Bar.

[0021] After obtaining the pretreated material, the present invention enzymatically hydrolyzes the pretreated material under vortex conditions, and after inactivating the enzyme, an enzymatic hydrolysis product is obtained.

[0022] In the present invention, the mass percentage of the alkaline protease is preferably 0.1‰ - 1.5‰, and can be 0.2‰ - 0.4‰, and can also be 0.3‰; the mass percentage of the neutral protease is preferably 0.4‰ - 1.2‰, can be 0.6‰ - 1.2‰, and can be 1.0‰; the mass percentage of the bromelain is preferably 0.1% - 0.5%, and can be 0.2%; the mass percentage of the papain is preferably 0.3‰ - 1.0‰, and can be 0.6‰. In the embodiments of the present invention, enzymatic hydrolysis is carried out by compounding 0.4‰ alkaline protease with 1.2‰ neutral protease; enzymatic hydrolysis is carried out by compounding 0.3‰ alkaline protease with 1.0‰ neutral protease and 0.2‰ bromelain; enzymatic hydrolysis by compounding 0.2‰ alkaline protease, 0.6‰ neutral protease and 0.6‰ papain is also included. The alkaline protease is derived from Bacillus subtilis with an activity of 300,000 U / ml. The neutral protease is derived from Bacillus subtilis with an activity of 120,000 U / ml. The papain is 800,000 U / g. The bromelain is 600,000 U / g.

[0023] In the present invention, the temperature of the protease enzymatic hydrolysis is preferably 55 - 65°C, and can also be 58 - 62°C, and can also be 60°C. The pH value of the system during the protease enzymatic hydrolysis is preferably 7.0 - 8.5, can be 7.5 - 8.0, and can also be 7.8. The reaction time of the protease enzymatic hydrolysis is 1 - 3 h, and can also be 2 - 2.5 h. The vortex rotation speed is preferably 60 rpm. The method for inactivating the enzyme preferably includes adjusting the pH value to acidic (4.5 - 4.8), and inactivating the enzyme by ultra-high pressure treatment or pulsed light treatment.

[0024] An enzymatic hydrolysis product is obtained. In the present invention, the liquid phase is separated from the enzymatic hydrolysis product and impurities are removed to obtain fish collagen peptides.

[0025] In the present invention, the method for separating the liquid phase from the enzymatic hydrolysate preferably uses a diaphragm press plate and frame filtration device for solid-liquid separation. The impurity removal preferably includes decolorization, re-deodorization, concentration and clarification. The decolorization preferably includes filtration through a filter membrane for decolorization. The pore size of the filter membrane is preferably 5000-20000 Da and can be 10000 Da. The method for re-deodorization preferably includes activated carbon adsorption. When using activated carbon adsorption, the working mass percentage of activated carbon is preferably 2%-4% and can be 3%. When using activated carbon adsorption, the temperature of the system is preferably 55-65 °C and can be 60 °C. The time for activated carbon adsorption is preferably 35-45 min and can be 40 min. The concentration method preferably includes concentrating with a 300 Dal nanofiltration membrane to 1 / 5 of the original volume, and then concentrating the obtained first concentrated solution to 35%-38% of the volume of the first concentrated solution by rotary evaporation to obtain a second concentrated solution. The clarification includes filtering the second concentrated solution with multi-layer fine cardboard and a PP filter element to collect the polypeptide concentrated solution.

[0026] In the present invention, after the impurity removal, it is preferred to sterilize the obtained polypeptide concentrated solution. The sterilization method preferably includes filtration through a filter membrane. The large pore size of the filter membrane is preferably 0.22 μm. The fish collagen peptide after sterilization preferably includes drying. The drying method includes spray drying. The inlet air temperature for spray drying is preferably 160-170 °C and can be 165 °C. The outlet air temperature for spray drying is preferably 90-95 °C and can be 92 °C. The atomization frequency for spray drying is preferably 20-25 Hz and can be 22 Hz.

[0027] The present invention provides a fish collagen peptide prepared by the preparation method. In the fish collagen peptide, the mass percentage of peptides with a molecular weight greater than 10000 Da is 0.01%-0.02%, the mass percentage of peptides with a molecular weight of 5000-10000 Da is 0.04%-0.83%, the mass percentage of peptides with a molecular weight of 3000-5000 Da is 0.68%-4.58%, the mass percentage of peptides with a molecular weight of 1000-3000 Da is 13.82%-34.42%; the mass percentage of peptides with a molecular weight of 500-1000 Da is 31.96%-32.76%, the mass percentage of peptides with a molecular weight of 189-500 Da is 24.15%-47.42%, and the mass percentage of peptides with a molecular weight less than 189 Da is 3.24%-5.89%.

[0028] In the present invention, in the fish collagen peptide, the mass percentage of peptides with a molecular weight greater than 10,000 Da is 0.01%, the mass percentage of peptides with a molecular weight of 5,000 - 10,000 Da is 0.67%, the mass percentage of peptides with a molecular weight of 3,000 - 5,000 Da is 4.25%, the mass percentage of peptides with a molecular weight of 1,000 - 3,000 Da is 32.76%; the mass percentage of peptides with a molecular weight of 500 - 1,000 Da is 32.76%, the mass percentage of peptides with a molecular weight of 189 - 500 Da is 26.58%, and the mass percentage of peptides with a molecular weight less than 189 Da is 3.77%.

[0029] In the present invention, scales and fish skins of different fish are used as materials respectively, and alkaline protease and neutral protease or their compound enzymes with bromelain or papain are used for enzymatic hydrolysis. The molecular weight of the prepared fish collagen peptide is mainly in the range of 189 - 3,000 Da. The protein content of the fish collagen peptide prepared in the present invention is above 99.23%, which is higher in protein purity than that of the fish collagen peptide extracted by conventional methods, and the molecular weight of the peptide is larger than that of the fish collagen peptide extracted by conventional methods.

[0030] The fish collagen peptide has the efficacy of brightening the skin and removing spots. The present invention provides the application of the fish collagen peptide in the preparation of drugs or skin care products for brightening the skin and removing spots, or in the preparation of health products, foods or food additives.

[0031] In the present invention, the types of spots removed include freckles, melasma, etc. The spot removal includes lightening spots and reducing the area of spots. The skin brightening includes improving skin dullness and brightening skin tone. In the embodiments of the present invention, the spot removal effect is evaluated by measuring the reduction in the area of skin spots, and the skin brightening effect is evaluated by the percentage increase in the L value and ITA value of the skin.

[0032] In the present invention, the product preferably includes at least one of the following: drugs, skin care products, health products, foods and food additives. The skin care products include face cream, body lotion, toner, facial mask, spot-removing essence, etc. The mass percentage of the fish collagen peptide in the skin care products is 1% - 20%, and can be 2%, 3%, 4%, 5%, 10% and 15%. The health products preferably include at least one of the following dosage forms: oral liquid, capsule, powder, granule and chewable tablet. The mass percentage of the fish collagen peptide in the health products is 5% - 95%, and can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50%, 60%, 70% and 80%. The foods include at least one of the following: beverages, meat products, fish products, egg products, pasta products, confectionery products and vegetable products. The mass percentage of the fish collagen peptide in the foods is 1% - 50%, and can be 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35% and 40%.

[0033] In one embodiment of the present invention, through human tests, taking the fish collagen peptide for 28 days can significantly improve the area of facial skin pigmentation in the subjects, indicating that the fish collagen peptide alone has the effect of removing skin pigmentation. At the same time, the present invention also measured the L value and ITA value of the facial skin of the subjects. The results showed that the fish collagen peptide can significantly increase the L value and ITA value of the facial skin of the subjects, indicating that the fish collagen peptide alone has the effect of improving skin brightness. It can be seen that the fish collagen peptide provided by the present invention has the effects of brightening the skin and removing skin pigmentation, and has a good effect on improving skin dullness, brightening skin gloss, inhibiting melanin deposition and reducing pigment accumulation.

[0034] The following is a detailed description of a fish collagen peptide with the effects of brightening the skin and removing skin pigmentation, its preparation method and application provided by the present invention in conjunction with embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0035] Example 1 Preparation method of a fish collagen peptide with the effects of brightening the skin and removing skin pigmentation (1) Rinsing: Fresh frozen grass carp scales are soaked in water for 20 h to thaw, and after draining, rinsed with water three times; (2) Degreasing and deodorizing: Add 3 times the mass of water to the rinsed fish scales, add chlorine dioxide preparation (food grade, ClO2 content 48%) with a final concentration of 0.8% and citric acid monohydrate (food grade, content > 99%) with a final concentration of 1.2%, and soak for 24 h. Stir intermittently, turn on for 5 min every 2 h, and after completion, rinse with water until the pH value is 6.8 and the conductivity is 467 μS / cm; (3) Mild heat treatment: Put the cleaned fish scales into the reaction tank, add 2.5 times the mass of water, heat up to 58 °C, and perform heat treatment for 40 min, with air blowing and stirring throughout the process; (4) Pulping: After the heat treatment is completed, the material is sheared at a high speed of 18,000 r / min for 45 s, homogenized at 22,000 rpm for 40 s, and then the homogenized slurry is transferred to a high-pressure homogenizer and homogenized in two stages at 1250 bar to obtain fish scale pulp material; (5) Enzymatic hydrolysis: The fish scale pulp material is in a vortex turbulent state under stirring at a speed of 30 rpm, and alkaline protease (derived from Bacillus subtilis, activity 300,000 U / ml) with a final concentration of 0.2‰, neutral protease (derived from Bacillus subtilis, activity 120,000 U / ml) with a final concentration of 0.6‰ and papain (800,000 U / g) with a final concentration of 0.6‰ are added. The enzymatic hydrolysis temperature is 58 °C, the pH value is 7.0 - 7.5, and the reaction is carried out for 2 h; (6) Inactivating enzymes: Adjust the pH value of the enzymatic hydrolysis product to 4.5, and inactivate the protease by pulsed intense light treatment for 5 s; (7) Primary clarification: Use a diaphragm press plate and frame filtration device (pressure 0.02 MPa) for solid-liquid separation to obtain a primary polypeptide solution; (8) Refinement: Pass the primary polypeptide solution through a 10,000 Dal membrane for decolorization. Add activated carbon at 3% of the solids content to the decolorized solution, adjust the pH value to 4.2, adsorb and remove fishy smell at 55 °C for 30 min. After completion, remove the activated carbon to obtain a refined polypeptide solution; (9) Concentration: Concentrate the refined polypeptide solution through a 300 Dal nanofiltration membrane to 20%. The first concentrated solution obtained is concentrated to 37% by a triple-effect evaporator (70 - 65 - 60 °C) to obtain a second concentrated solution; (10) Secondary clarification: Filter the second concentrated solution with multi-layer fine cardboard and PP filter elements (cardboard pore size 5 μm, filter element pore sizes 0.45 and 0.22 μm) to obtain a clarified polypeptide concentrated solution; (11) Sterilization: Filter and sterilize the clarified polypeptide concentrated solution with a 0.22 μm microporous filter element to obtain a sterilized polypeptide concentrated solution; (12) Drying: Spray-dry the sterilized polypeptide concentrated solution, with an inlet air temperature of 160 °C, an outlet air temperature of 90 °C, and an atomization frequency of 25 Hz to obtain fish collagen peptide.

[0036] Determine the components of the prepared fish collagen peptide, and determine the content ratio of peptides in each molecular weight range (GB / T 31645 - 2008).

[0037] The product test results are shown in Table 1.

[0038] Table 1 Fish collagen peptide from grass carp scales

[0039] Example 2 A preparation method of fish collagen peptide with skin brightening and freckle removing effects (1) Rinsing: Soak fresh-frozen black fish scales in water for 18 h for thawing. After draining, add clean water and rinse 3 times; (2) Degreasing and deodorizing: Add 3 times the mass of clean water to the rinsed fish scales, and then add hydrogen peroxide (food grade, mass fraction 32 - 36%) with a final concentration of 0.2% and sodium hydroxide (liquid, mass fraction 30 - 33%) with a final concentration of 0.3%. Soak for 24 h. Stir intermittently, stir for 5 min every 2 h. After completion, rinse with clean water until the pH value is 7.2 and the conductivity is 386 μS / cm; (3) Mild heat treatment: Put the cleaned fish scales into a reaction tank, add 2 times the mass of clean water, heat up to 65 °C, and perform heat treatment for 60 min. Keep air blowing and stirring throughout the process; (4)Pulping: After heat treatment, the material is sheared at a high speed of 15,000 r / min for 50 s, emulsified at 2,500 r / min, and homogenized at a high speed in two stages under a pressure of 1,400 Bar to obtain a scaly pulping material; (5)Enzymolysis: The scaly fish scale pulping material is in a vortex turbulent state under ultra-high speed stirring. Add alkaline protease (from Bacillus subtilis, activity 350,000 U) with a final concentration of 0.3‰, neutral protease (from Bacillus subtilis, activity 150,000 U) with a concentration of 1.0‰, and bromelain (600,000 U) with a concentration of 0.2‰. The temperature is 60 °C, the pH is controlled at 7.5 - 7.8, and the reaction lasts for 2.5 h; (6)Inactivation of enzymes: After the reaction, the pH value of the system is adjusted to 4.5, and the protease is inactivated by pulsed intense light treatment for 5 s; (7)Primary clarification: Use a diaphragm press plate and frame filtration device for solid-liquid separation to obtain a primary polypeptide solution; (8)Refining: The primary polypeptide solution is decolorized through a 10,000 Dal membrane, then activated carbon with 2% of the solid content is added, and at the same time the pH value is adjusted to 4.5, and it is adsorbed and deodorized at 60 °C for 40 min. After the reaction, the activated carbon is removed to obtain a refined polypeptide solution; (9)Concentration: The refined polypeptide solution is concentrated to 20% through a 300 Dal nanofiltration membrane. The obtained first concentrated solution is then concentrated to 38% through a triple-effect evaporator (70 - 65 - 60 °C) to obtain a second concentrated solution; (10)Secondary clarification: The second concentrated solution is filtered with multi-layer fine cardboard and PP filters (the pore size of the cardboard is 5 μm, and the pore sizes of the filters are 0.45 and 0.22 μm) to obtain a clarified polypeptide concentrated solution; (11)Sterilization: Sterilize by filtering with a 0.22 μm microporous filter element to obtain the sterilized polypeptide; (12)Drying: Spray dry the sterilized polypeptide, with an inlet air temperature of 170 °C, an outlet air temperature of 95 °C, and an atomization frequency of 28 Hz to obtain a fish collagen peptide product derived from black fish scales.

[0040] Detect the fish collagen peptide product derived from black fish scales according to the method of Example 1. The product detection results are shown in Table 2.

[0041] Table 2 Determination results of fish collagen peptide derived from black fish scales

[0042] Example 3 A preparation method of fish collagen peptide with skin brightening and freckle removing effects (1)Rinsing: Fresh frozen cod skin is thawed by soaking in water for 24 h, drained, and then rinsed 3 times with clean water; (2)Degreasing and removing fishy smell: Add 4 times the mass of clear water, 2.0% chlorine dioxide preparation (food grade, ClO2 content 48%) and 2.5% malic acid (food grade, content > 99%) to the fish skin, and soak for 18 h. Stir intermittently, turn on for 2 min every 6 h, and after completion, rinse with clear water until the pH is 6.8 and the conductivity is 479 μS / cm; (3)Mild heat treatment: After crushing the cleaned fish skin, put it into a reaction tank, add 1.5 times the mass of clear water, heat up to 60 °C, and perform heat treatment for 30 min, with air blowing and stirring throughout the process; (4)Pulping: After the heat treatment is completed, the material is emulsified by high-speed shearing (10000 r / min) and high-pressure homogenized to obtain fish skin pulp material; (5)Enzymolysis: Keep the fish skin pulp material in a vortex turbulent state under ultra-high-speed stirring, add 0.4‰ alkaline protease (derived from Bacillus subtilis, activity 250,000 U) and 1.2‰ neutral protease (derived from Bacillus subtilis, activity 100,000 U), at a temperature of 60 °C, control the pH at 7.5 - 8.0, and react for 3 h; (6)Inactivating enzymes: After completion, adjust the pH of the system to 4.8, and inactivate the protease by ultra-high pressure treatment; (7)Primary clarification: Use a diaphragm press plate and frame filtration equipment for solid-liquid separation to obtain a primary polypeptide solution; (8)Refining: Pass the polypeptide solution through a 10000 Dal membrane for decolorization, then add activated carbon with 5% solids content for adsorption and deodorization, control the pH at 4.8, the temperature at 65 °C, and the time at 60 min. After completion, remove the activated carbon to obtain a refined polypeptide solution; (9)Concentration: Concentrate to 20% through a 300 Dal nanofiltration membrane, and then concentrate to 35% through a triple-effect evaporator (70 - 65 - 60 °C); (10)Secondary clarification: Filter the concentrated solution with multi-layer fine cardboard and PP filter elements to obtain a clarified polypeptide concentrated solution; (11)Sterilization: Filter and sterilize with a 0.22 μm microporous filter element; (12)Drying: Spray drying, inlet air temperature 165 °C, outlet air 92 °C, atomization frequency 22 Hz, to obtain fish collagen peptide derived from cod skin.

[0043] Detect the fish collagen peptide product according to the method of Example 1, and the product detection results are shown in Table 3.

[0044] Table 3 Detection results of fish collagen peptide derived from cod skin

[0045] Example 4 Human experiment on freckle removal and skin brightening The product prepared in Example 1 was subjected to a human trial to evaluate its efficacy in removing freckles and brightening the skin. A total of 31 subjects were enrolled in this experiment, numbered from 1 to 31. Among them, the subject numbered 13 withdrew from the trial for personal reasons, and the effective number of test subjects was 30, with an average age of (45.0 ± 9.2) years. The human skin test experiment of fish collagen peptide was specifically entrusted to Hubei Ruibang Biotechnology Co., Ltd. for testing, with the number BSEYNNUB4537357R7. The method of taking fish collagen peptide is one pack each time, stir evenly with warm water and take it, twice a day, once in the morning and once in the evening, and the taking cycle is 28 days. The Wilcoxon paired rank sum test method was used to statistically analyze the basic value and the measurement results after taking the sample for 28 days for the measurement results of the freckle area.

[0046] The results are shown in Table 4 and Figure 1 . Compared with the basic value, the freckle area was significantly reduced after taking the sample for 28 days ( P = 0.0013).

[0047] Table 4 Test results of the proportion of freckle area of the subjects

[0048] The paired sample T test was used to statistically analyze the measured skin L value results of the subjects. The results are shown in Table 5 and Figure 2 . Compared with the basic value, the skin L value of the subjects increased by 2.19% after taking the sample for 28 days, and there was a significant difference ( P <0.001).

[0049] Table 5 Test results of the skin L value of the subjects

[0050] The paired Wilcoxon paired rank sum test method was used to statistically analyze the measured skin ITA results of the subjects. The results are shown in Table 6 and Figure 3 . Compared with the basic value, the skin ITA value of the subjects increased by 9.19% after taking the sample for 28 days, and there was a significant difference ( P <0.001).

[0051] Table 6 Test results of the skin ITA value of the subjects

[0052] Comparative Example 1 A preparation method of fish collagen peptide (1) Rinsing: Fresh frozen grass carp scales were soaked in clear water for 20 h to thaw, and after draining, they were rinsed 3 times with clear water; (2) Depickling and degreasing and removing fishy smell: Scale deashing: Add 4 times the mass of clear water and 5% food-grade hydrochloric acid (37% mass fraction) to the rinsed fish scales, soak and react for 18 h. This process is carried out in a special reactor for scale deashing, with stirring throughout, monitoring the pH value of the system (controlled below 1.0), and after completion, rinse with clear water until the pH value reaches 5.5; Degreasing and deodorization: Add 3 times the mass of clear water, 8% hydrogen peroxide (food-grade, 35% H2O2 content) and 5% liquid alkali (food-grade, 32% content) to the fish scales, soak for 24 h. Stir intermittently, stir for 5 min every 2 h, and after completion, rinse with clear water until the pH value reaches 8.0; (3) Heat treatment: Heat to 95 °C and maintain for 2 h; (4) Enzymatic hydrolysis: Cool the heat-treated material to 60 °C, add alkaline protease with a final concentration of 2‰ (derived from Bacillus subtilis, 350,000 U), control the pH value at 9.0 - 9.5, and carry out enzymatic hydrolysis for 2.5 h to obtain an enzymatic hydrolysis product; (6) Enzyme inactivation: Heat the enzymatic hydrolysis product to 95 °C and maintain for 30 min; (7) Primary clarification: Filter through a plate and frame filter, collect the filtrate to obtain an enzymatic hydrolysate; (8) Refinement: Add 10% activated carbon based on the solids content to the enzymatic hydrolysate, decolorize at pH 4.5 and 85 °C for 1.5 h to obtain a refined polypeptide solution; (9) Concentration: Concentrate the refined polypeptide solution to 25% through a nanofiltration membrane to obtain a first concentrated solution, and then concentrate it to 45% using a scraper evaporator to obtain a second concentrated solution; (10) Secondary clarification: Filter the second concentrated solution through cardboard to obtain a clarified solution; (11) Sterilization: Pass the clarified solution through a 0.22 μm microporous filter element to obtain a sterilized polypeptide solution; (12) Drying: Spray-dry the sterilized polypeptide solution, with an inlet air temperature of 195 °C, an outlet air temperature of 95 °C, and an atomization frequency of 25 Hz to obtain fish collagen peptides derived from grass carp scales.

[0053] Measure the fish collagen peptides derived from grass carp scales prepared above according to the method of Example 1, and the measurement results are shown in Table 7.

[0054] Table 7 Measurement results of fish collagen peptides derived from grass carp scales prepared by the traditional method

[0055] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing fish collagen peptide, characterized in that, It includes the following steps: Subject the defatted and deodorized fish scales to heat treatment and homogenization treatment to obtain a pretreated material; The method for defatting and deodorizing the fish scales includes soaking treatment with an oxidant and an acid or a base simultaneously; Subject the pretreated material to protease hydrolysis under vortex conditions, and after inactivating the enzyme, obtain a hydrolysate; The protease includes at least two of alkaline protease, neutral protease, bromelain and papain; Separate the liquid phase from the hydrolysate, remove impurities, and obtain fish collagen peptides.

2. The preparation method according to claim 1, characterized in that, The pH value of the defatted and deodorized fish scales is 6.5 - 7.5; The conductivity of the defatted and deodorized fish scales < 500 μS / cm.

3. The preparation method according to claim 2, characterized in that, The oxidant includes an oxidant with any of the following working concentrations: chlorine dioxide preparation with a volume concentration of 0.6% - 2.0%, hydrogen peroxide with a volume concentration of 0.1% - 0.5%, and ozone with a concentration of 0.1 - 0.15 ppm; The acid includes an aqueous solution of citric acid or malic acid with a mass percentage of 0.9% - 1.5% or an aqueous solution of hydrochloric acid with a volume percentage of 0.1% - 0.3%; The base includes an aqueous solution of sodium hydroxide with a mass percentage of 0.1% - 0.3%.

4. The preparation method according to claim 1, characterized in that, The temperature of the heat treatment is 55 - 100 °C; the time of the heat treatment is 30 - 90 min.

5. The preparation method according to claim 1, wherein The homogenization treatment includes high-speed shearing, emulsification, homogenization and homogenizing; The rotation speed of the high-speed shearing is 10000 - 18000 rpm; the time of the high-speed shearing is 30 - 60 s; The speed of the emulsification is 2000 - 2500 r / min, and the time of the emulsification is 20 - 30 min; The rotation speed of the homogenization is 20000 - 25000 r / min, and the time of the homogenization is 30 - 60 s; The pressure of the homogenizing is 1000 - 1500 bar.

6. The preparation method according to claim 1, wherein In the hydrolysis system, the mass percentage of the alkaline protease is 0.1‰ - 1.5‰; The mass percentage of the neutral protease is 0.4‰ - 1.2‰; The mass percentage of the bromelain is 0.1% - 0.5%; The mass percentage of the papain is 0.3‰ - 1.0‰; The temperature of the protease hydrolysis is 55 - 65 °C; The pH value of the system during protease hydrolysis is 7.0 - 8.5; The reaction time of the protease hydrolysis is 1 - 3 h.

7. The preparation method according to any one of claims 1 to 6, characterized in that, The impurity removal includes decolorization and re-deodorization; The decolorization includes decolorization by membrane filtration; the pore size of the membrane is 5000 - 20000 Da; The method for re-deodorization includes activated carbon adsorption.

8. The fish collagen peptide prepared by the preparation method according to any one of claims 1 to 7, characterized in that, In the fish collagen peptide, the mass percentage of peptides with a molecular weight greater than 10,000 Da is 0.01% - 0.02%, the mass percentage of peptides with a molecular weight of 5,000 - 10,000 Da is 0.04% - 0.83%, the mass percentage of peptides with a molecular weight of 3,000 - 5,000 Da is 0.68% - 4.58%, the mass percentage of peptides with a molecular weight of 1,000 - 3,000 Da is 13.82% - 34.42%; the mass percentage of peptides with a molecular weight of 500 - 1,000 Da is 31.96% - 32.76%, the mass percentage of peptides with a molecular weight of 189 - 500 Da is 24.15% - 47.42%, and the mass percentage of peptides with a molecular weight less than 189 Da is 3.24% - 5.89%.

9. Use of the fish collagen peptide according to claim 8 in the preparation of a drug or skin care product for brightening the skin and removing freckles, or in the preparation of a health product, food or food additive.

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